Modified VEGF Oligonucleotides for Inhibition of tumor growth

ABSTRACT

Disclosed are oligonucleotides complementary to VEGF-specific nucleic acid useful in reducing the expression of VEGF. Also disclosed are pharmaceutical formulations containing such oligonucleotides useful for treating various disorders associated with neovascularization and angiogenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/629,730, entitled “MODIFIED VEGF OLIGONUCLEOTIDES FOR THETREATMENT OF SKIN DISORDERS,” filed Apr. 9, 1996 (abandoned), which is acontinuation-in-part of U.S. patent application Ser. No. 08/569,926,entitled “MODIFIED VEGF OLIGONUCLEOTIDES,” filed Dec. 8, 1995 (U.S. Pat.No. 5,641,756).

BACKGROUND OF THE INVENTION

This invention relates to vascular endothelial growth factor. Morespecifically, this invention relates to oligonucleotides specific forvascular endothelial growth factor nucleic acid and useful treatment ofdisorders that are associated with neovascularization and angiogenesis,such as psoriasis.

Neovascular diseases of the retina such as diabetic retinopathy,retinopathy of prematurity, and age-related macular degeneration are amajor cause of blindness in the United States and the world, yet thebiochemical events responsible for these processes have not been fullyelucidated.

Diabetic retinopathy is the leading cause of blindness among working ageadults (20-64) in the United States (Foster in Harrison's Principles ofInternal Medicine (Isselbacher et al., eds.) McGraw-Hill, Inc., New York(1994) pp. 1994-1995). During the course of diabetes mellitus, theretinal vessels undergo changes that result in not only leaky vesselsbut also vessel drop out resulting in retinal hypoxia. The effects ofthese complications are hemorrhaging, “cotton wool” spots, retinalinfarcts, and neovascularization of the retina resulting in bleeding andretinal detachment. If left untreated, there is a 60% chance of visualloss. Classic treatment for proliferative diabetic retinopathy ispanretinal laser photocoagulation (PRP). However, complications canoccur from panretinal laser photocoagulation such as foveal burns,hemorrhaging, retinal detachment, and choroidal vessel growth.Furthermore, other untoward effects of this treatment are decreasedperipheral vision, decreased night vision, and changes in colorperception (Am. J. Ophthamol. (1976) 81:383-396; Ophthalmol. (1991)98:741-840). Thus, there is a need for a more effective treatment fordiabetic retinopathy.

Retinopathy of prematurity (ROP) is a common cause of blindness inchildren in the United States (Pierce et al. (1994) Int. Ophth. Clinics34:121-148). Premature babies are exposed to hyperoxic conditions afterbirth even without supplemental oxygen because the partial pressure ofoxygen in utero is much lower than what is achieved when breathingnormal room air. This relative hyperoxia is necessary for their survivalyet can result in ROP. The blood vessels of the retina cease to developinto the peripheral retina resulting in ischemia and localized hypoxicconditions as the metabolic demands of the developing retina increase.The resulting hypoxia stimulates the subsequent neovascularization ofthe retina. This neovascularization usually regresses but can lead toirreversible vision loss. There are at least 10,000 new-cases per yearwith a worldwide estimate of 10 million total cases. At present, thereis no effective cure for ROP. Two therapeutic methods, cryotherapy andlaser therapy, have been used but are not completely effective andthemselves cause damage to the eye, resulting in a reduction of vision(Pierce et al. (1994) Int. Ophth. Clinics 34:121-148). Many otherantiangiogenic compounds have been tested, but no inhibition in retinalneovascularization has been reported (Smith et al. (1994) Invest.Ophthamol. Vis. Sci. 35:1442; Foley et al. (1994) Invest. Ophthamol.Vis. Sci. 35:1442). Thus, there is a need for an effective treatment forROP.

Age related macular degeneration is one of the leading causes ofblindness in older adults in the United States, and may account for upto 30% of all bilateral blindness among Caucasian Americans (Anonymous(1994) Prevent Blindness America). This disease is characterized by lossof central vision, usually in both eyes, due to damage to retinalpigment epithelial cells which provide physiological support to thelight sensitive photoreceptor cells of the retina. In most cases thereis currently no effective treatment. In approximately 20% of exudativecases that are diagnosed early, laser treatment can prevent further lossof vision; however, this effect is temporary (Bressler et al.,Principles and Practices of Ophthamology (eds. Albert and Jakobiac),W.B. Saunders Co., Philadelphia, Pa.) (1994) Vol. 2 pp. 834-852). Thus,there is a need for a more effective and permanent treatment for agerelated macular degeneration.

Ocular neovascularization is also the underlying pathology in sicklecell retinopathy, neovascular glaucoma, retinal vein occlusion, andother hypoxic diseases. These eye diseases as well as other pathologicalstates associated with neovascularization (i.e., tumor growth, woundhealing) appear to have hypoxia as a common factor (Knighton et al.(1983) Science 221:1283-1285; Folkman et al. (1987) Science 235:442-446;Klagsbrun et al. (1991) Ann. Rev. Physiol. 53:217-239; Miller et al.(1993) Principles and Practice of Ophthamology, W.B. Saunders,Philadelphia, pp. 760; and Aiello et al. (1994) New Eng. J. Med.331:1480-1487). Moreover, retinal neovascularization has beenhypothesized to be the result of a “vasoformative factor” which isreleased by the retina in response to hypoxia (Michaelson (1948) Trans.Ophthamol. Soc. U. K. 68:137-180; and Ashton et al. (1954) Br. J.Ophthamol. 38:397-432). Recent experimental data show a high correlationbetween vascular endothelial growth factor expression and retinalneovascularization (Aiello et al. (1994) New Eng. J. Med.331:1480-1487). Furthermore, elevated levels of vascular endothelialgrowth factor have recently been found in vitreous from patients withdiabetes (Aiello et al., ibid.). Thus, this cytokine/growth factor mayplay an important role in neovascularization-related disease.

Vascular endothelial growth factor/vascular permeability factor(VEGF/VPF) is an endothelial cell-specific mitogen which supportsangiogenesis in wound healing and development. In addition, VEGF hasrecently been shown to be stimulated by hypoxia and required for tumorangiogenesis (Senger et al. (1986) Cancer 46:5629-5632; Kim et al.(1993) Nature 362:841-844; Schweiki et al. (1992) Nature 359:843-845;Plate et al. (1992) Nature 359:845-848). It is a 34-43 kD (with thepredominant species at about 45 kD) dimeric, disulfide-linkedglycoprotein synthesized and secreted by a variety of tumor and normalcells. In addition, cultured human retinal cells such as pigmentepithelial cells and pericytes have been demonstrated to secrete VEGFand to increase VEGF gene expression in response to hypoxia (Adamis etal. (1993) Biochem. Biophys. Res. Commun. 193:631-638; Plouet et al.(1992) Invest. Ophthamol. Vis. Sci. 34:900; Adamis et al. (1993) Invest.Ophthamol. Vis. Sci. 34:1440; Aiello et al. (1994) Invest. Ophthamol.Vis. Sci. 35:1868; Simorre-Pinatel et al. (1994) Invest. Ophthamol. Vis.Sci. 35:3393-3400). In contrast, VEGF in normal tissues is relativelylow.

VEGF has also been shown to play a major role in other diseasesassociated with the aberrant angiogenesis, including tumor developmentand skin disorders. Conditions associated with such irregularitiesinclude cancer, rheumatoid arthritis, the bullous diseases (includingbullous pemphigoid, dermatitis herpetiformis, and erythema multiforme),and psoriasis.

Psoriasis is a chronic skin disorder that affects one in fifty peopleworld wide and over five million people in the United States.Approximately 150,000 to 250,000 new cases of psoriasis are diagnosedeach year. Ten percent of people with psoriasis develop psoriaticarthritis. The most common form of the disease is called plaquepsoriasis or psoriasis vulgaris. Other forms are pustular, guttate,inverse, and erythrodermic psoriasis.

The cause of psoriasis is unknown. The skin lesions of psoriasisvulgaris are in part a result of an excessive rapid growth and turnoverof keratinocytes. Current types of treatment achieve some temporaryrelief (e.g., steroids, anthralin, calcipotriene, coal tar with orwithout light therapy, psoralen with UVA treatment, methotrexate,retinoids with or without UV light, and cyclosporin A). However, harmfulside effects of these treatments exist which vary in degree of healthhazard, and some must be carefully monitored by a physician. Furthermoremost of these treatments result in a recurrence of the psoriasicsymptoms.

Ballaun et al. (J. Invest. Dermatol. (1995) 104:7-10) have shown thatthe three major splice forms of VEGF are produced by human epidermalkeratinocytes and can be detected in the supernatant of cell cultures.In addition, VEGF upregulation has been observed in bullous pemphigoid,dermatitis herpetiformis, and erythema multiforme (Brown et al. (1995)J. Invest Dermatol. 104:744-749). Furthermore, psoriasic lesions expresselevated levels of transforming growth factor alpha (TGFα), a knowninducer of VEGF in cell culture (Detmar et al. (J. Invest. Dermatol.(1995) 105:44-50), and VEGF levels of normal epidermal keratinocytes canbe induced in response to elevated levels of TGFα.

Thus, VEGF appears to play a principle role in many pathological statesand processes related to angiogenesis and neovascularization. Regulationof VEGF expression in tissues affected by the various conditionsdescribed above could therefore be key in treatment or preventativetherapies associated with such disorders.

New chemotherapeutic agents termed “antisense oligonucleotides” havebeen developed which are capable of modulating cellular and foreign geneexpression (see, Zamecnik et al. (1978) Proc. Natl. Acad. Sci. (USA)75:280-284). Without being limited to any theory or mechanism, it isgenerally believed that the activity of antisense oligonucleotidesdepends on the binding of the oligonucleotide to the target nucleic acid(e.g. to at least a portion of a genomic region, gene or mRNA transcriptthereof), thus disrupting the function of the target, either byhybridization arrest or by destruction of target RNA by RNase H (theability to activate RNase H when hybridized to RNA).

VEGF-specific antisense oligonucleotides have been developed (Uchida etal. (1995) Antisense Res. & Dev. 5(1):87 (Abstract OP-10); Nomura etal., (1995) Antisense Res. & Dev. 5(1):91 (Abstract OP-18)), althoughnone have been demonstrated to reverse neovascularization orangiogenesis. Thus, a need still remains for the development ofoligonucleotides that are capable of reducing VEGF expression, andultimately, of inhibiting the onset of diseases and disorders associatedwith the expression of VEGF.

SUMMARY OF THE INVENTION

It is known that cells affected by hypoxia induce VEGF, and thataberrant expression of VEGF has been observed in skin diseasescharacterized by neoangiogenesis and epidermal alterations. The presentinvention provides novel synthetic oligonucleotides specific fornucleotides 58 to 90 of the VEGF gene which can reduce the hypoxia- orTGFα-induced expression of VEGF mRNA and protein. This information hasbeen exploited to develop the present invention which includesVEGF-specific oligonucleotides, pharmaceutical formulations, and methodsof reducing the expression of VEGF mRNA and protein and of treatingvarious diseases characterized by the over-expression of VEGF.

In one aspect, the invention provides a synthetic oligonucleotidecomplementary to a nucleic acid specific for human vascular endothelialgrowth factor. This oligonucleotide has a nucleic acid sequence setforth in the Sequence Listing as SEQ ID NOS:1-16.

As used herein, the term “synthetic oligonucleotide” refers tochemically synthesized polymers of nucleotides covalently attached viaat least one 5′ to 3′ internucleotide linkage. In some embodiments,these oligonucleotides contain at least one deoxyribonucleotide,ribonucleotide, or both deoxyribonucleotides and ribonucleotides. Inanother embodiment, the synthetic oligonucleotides used in the methodsof the invention are from about 15 to about 30 nucleotides in length. Inpreferred embodiments, these oligonucleotides contain from about 16 to29 nucleotides.

For purposes of the invention, the term “oligonucleotide sequence thatis complementary to a genomic region or an RNA molecule transcribedtherefrom” is intended to mean an oligonucleotide that binds to thenucleic acid sequence under physiological conditions, e.g., byWatson-Crick base pairing (interaction between oligonucleotide andsingle-stranded nucleic acid) or by Hoogsteen base pairing (interactionbetween oligonucleotide and double-stranded nucleic acid) or by anyother means including in the case of a oligonucleotide binding to RNA,causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen basepairing under physiological conditions is measured as a practical matterby observing interference with the function of the nucleic acidsequence.

In some embodiments, the synthetic oligonucleotide of the invention aremodified in a number of ways without compromising their ability tohybridize to nucleotide sequences contained within the mRNA for VEGF.The term “modified oligonucleotide” as used herein describes anoligonucleotide in which at least two of its nucleotides are covalentlylinked via a synthetic linkage, i.e., a linkage other than aphosphodiester linkage between the 5′ end of one nucleotide and the 3′end of another nucleotide in which the 5′ nucleotide phosphate has beenreplaced with any number of chemical groups. In some preferredembodiments, at least one internucleotide linkage of the oligonucleotideis an alkylphosphonate, phosphorothioate, phosphorodithioate, phosphateester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate,phosphate triester, acetamidate, and/or carboxymethyl ester.

The term “modified oligonucleotide” also encompasses oligonucleotideshaving at least one nucleotide with a modified base and/or sugar, suchas a 2′-O-substituted ribonucleotide. For purposes of the invention, theterm “2′-O-substituted” means substitution of the 2′ position of thepentose moiety with an —O— lower alkyl group containing 1-6 saturated orunsaturated carbon atoms, or with an —O-aryl or allyl group having 2-6carbon atoms, wherein such alkyl, aryl or allyl group may beunsubstituted or may be substituted, e.g., with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halogroup, but not with a 2′-H group. In some embodiments theoligonucleotides of the invention include four or five ribonucleotides2′-O-alkylated at their 5′ terminus (i.e., 5′ 2-O-alkylatedribonucleotides), and/or four or five ribonucleotides 2′-O-alkylated attheir 3′ terminus (i.e., 3′ 2-O-alkylated ribonucleotides). In preferredembodiments, the nucleotides of the synthetic oligonucleotides arelinked by a or at least one phosphorothioate internucleotide linkage.The phosphorothioate linkages may be mixed R_(p) and S_(p) enantiomers,or they may be stereoregular or substantially stereoregular in eitherR_(p) or S_(p) form (see Iyer et al. (1995) Tetrahedron Asymmetry6:1051-1054).

In another aspect, the invention provides a method of inhibiting VEGFexpression. In this method, nucleic acid specific for VEGF is contactedwith an oligonucleotide of the invention. As used herein, the term“nucleic acid” encompasses a genomic region or an RNA moleculetranscribed therefrom. In some embodiments, the nucleic acid is mRNA.

Without being limited to any theory or mechanism, it is generallybelieved that the activity of oligonucleotides used in accordance withthis invention depends on the hybridization of the oligonucleotide tothe target nucleic acid (e.g. to at least a portion of a genomic region,gene or mRNA transcript thereof), thus disrupting the function of thetarget. Such hybridization under physiological conditions is measured asa practical matter by observing interference with the function of thenucleic acid sequence. Thus, a preferred oligonucleotide used inaccordance with the invention is capable of forming a stable duplex (ortriplex in the Hoogsteen pairing mechanism) with the target nucleicacid; activate RNase H thereby causing effective destruction of thetarget RNA molecule, and in addition is capable of resisting nucleolyticdegradation (e.g. endonuclease and exonuclease activity) in vivo. Anumber of the modifications to oligonucleotides described above andothers which are known in the art specifically and successfully addresseach of these preferred characteristics.

Also provided by the present invention is a pharmaceutical compositioncomprising at least one synthetic oligonucleotide of claim 1 in aphysiologically acceptable carrier.

Another aspect of the invention includes pharmaceutical compositionscapable of inhibiting neovascularization and thus are useful in themethods of the invention. These compositions include a syntheticoligonucleotide of the present invention which specifically inhibits theexpression of vascular endothelial growth factor and a physiologicallyand/or pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredient(s). The term “physiologically acceptable” refersto a non-toxic material that is compatible with a biological system suchas a cell, cell culture, tissue, or organism.

Another aspect of the invention is assessment of the role of VEGF inneovascularization and angiogenesis associated with normal developmentand in various disease states.

Yet another aspect is a method of treating psoriasis by administering toa human afflicted with the disorder a therapeutically-affective amountof an oligonucleotide of the invention.

The subject oligonucleotides and methods of the invention also provide ameans of examining the function of the VEGF gene in a cell, or in acontrol mammal and in a mammal afflicted with aneovascularization-related or psoriasis-related disorder. The cell ormammal is administered the oligonucleotide, and the expression of VEGFmRNA or protein and/or proteins which are known to interact with CDK4 isexamined. Presently, gene function is often examined by the arduous taskof making a “knock out” animal such as a mouse. This task is difficult,time-consuming and cannot be accomplished for genes essential to animaldevelopment since the “knock out” would produce a lethal phenotype. Thepresent invention overcomes the shortcomings of this model.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present invention, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings in which:

FIG. 1 is a schematic representation of the regions of the VEGF cDNAsequence that are targeted by oligonucleotides of the invention;

FIG. 2 is a graphic representation of ELISA results demonstrating theability of oligonucleotides H3-I and H3-J to inhibit VEGF expressioninduced by cobalt chloride (CoCl₂);

FIG. 3 is a graphic representation of ELISA results demonstrating theability of oligonucleotides H3-D H3-E, H3-F to inhibit VEGF expressioninduced by cobalt chloride;

FIG. 4 is a graphic representation of ELISA results demonstrating theability of oligonucleotides H3-G and H3-H to inhibit VEGF expressioninduced by cobalt chloride;

FIG. 5 is a graphic representation of ELISA results demonstrating theability of oligonucleotides H3 and H3-I to inhibit VEGF expressioninduced by cobalt chloride in M21 human melanoma cells in vitro;

FIG. 6 is a graphic representation of ELISA results demonstrating theability of modified H3 oligonucleotides to inhibit VEGF expressioninduced by cobalt chloride (H3-K: all 2′-O-methylated phosphorothioateribonucleotides; H3-L: five 5′ 2′-O-alkylated phosphorothioateribonucleotides, the remainder, phosphorothioate deoxyribonucleotides;H3-M: five 3′ 2′-O-alkylated phosphorothioate ribonucleotides; theremainder, phosphorothioate deoxyribonucleotides; and R3-N: five 3′2′-O-alkylated phosphorothioate ribonucleotides, five 5′ 2′-O-alkylatedphosphorothioate ribonucleotides, and the remainder, phosphorothioatedeoxyribonucleotides);

FIG. 7 is a graphic representation of the results of an ELISA in whichthe expression of TGF-induced VEGF in human keratinocytes in thepresence and absence of oligonucleotides of the invention is shown;

FIG. 8 is a graphic representation of the results of a Northern blotanalysis showing the ability of PS oligonucleotides of the invention toinhibit expression of VEGF mRNA in NHEK cells 24 and 48 hours afteroligonucleotide treatment; and

FIG. 9 is a graphic representation of the results of a capture ELISAshowing the ability of PS oligonucleotides of the invention to inhibitexpression of VEGF protein in NHEK cells 24, 48, 72, 96, and 120 hoursafter oligonucleotide treatment;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. The issuedU.S. patents, allowed applications, and references cited herein arehereby incorporated by reference.

The present invention provides synthetic antisense oligonucleotidesspecific for VEGF nucleic acid which are useful in treating diseases anddisorders associated with angiogenesis and neovascularization, includingretinal neovascularization, psoriasis, and the bullous diseases.

Antisense oligonucleotide technology provides a novel approach to theinhibition of gene expression (see generally, Agrawal (1992) Trends inBiotech. 10:152-158; Wagner (1994) Nature 372:333-335; and Stein et al.(1993) Science 261:1004-1012). By binding to the complementary nucleicacid sequence (the sense strand), antisense oligonucleotide are able toinhibit splicing and translation of RNA. In this way, antisenseoligonucleotides are able to inhibit protein expression. Antisenseoligonucleotides have also been shown to bind to genomic DNA, forming atriplex, and inhibit transcription. Furthermore, a 17mer base sequencestatistically occurs only once in the human genome, and thus extremelyprecise targeting of specific sequences is possible with such antisenseoligonucleotides.

It has been determined that the VEGF coding region is comprised of eightaxons (Tischer et al. (1994) J. Biol. Chem. 266:11947-11954). Three VEGFtranscripts, 121, 165, and 189 amino acids long, have been observed,suggesting that an alternative splicing mechanism is involved (Leung etal. (1989) Science 246:1306-1309; Tischer et al. (1991) J. Biol. Chem.266:11947-11954). More recently, a fourth VEGF transcript was discoveredwhich has a length encoding 206 amino acids (Houck et al. (1991) Mol.Endocrinol. 5:1806-1814). Transcripts analogous to the 121 and 165 aminoacid polypeptides have been identified in the bovine system (Leung etal. (1989) Science 246:1306-1309), and the transcript corresponding tothe 165 amino acid transcript have also been identified in the rodentsystem (Conn et al. (1990) Proc. Natl. Acad. Sci. (USA) 87:1323-1327);Senger et al. (1990) Cancer Res. 50:1774-1778; Claffey et al. (1992) J.Biol. Chem. 267:16317-16322). Nucleic acid sequences encoding threeforms of VEGF have also been reported in humans (Tischer et al. (1991)J. Biol. Chem. 266:11947-11954), and comparisons between the human andthe murine VEGF have revealed greater than 85% interspecies conservation(Claffey et al. (1992) J. Biol. Chem. 267:16317-16322).

The oligonucleotides of the invention are directed to any portion of theVEGF nucleic acid sequence that effectively acts as a target forinhibiting VEGF expression. The sequence of the gene encoding VEGF hasbeen reported in mice (Claffey et al., ibid.) and for humans (Tischer etal., ibid.). These targeted regions of the VEGF gene include anyportions of the known exons. In addition, exon-intron boundaries arepotentially useful targets for antisense inhibition of VEGF expression.One useful targeted region is around bases 58 to 90. The nucleotidesequences of some representative, non-limiting oligonucleotides specificfor human VEGF have SEQ ID NOS:1-16.

The oligonucleotides of the invention are composed of ribonucleotides,deoxyribonucleotides, or a combination of both, with the 5′ end of onenucleotide and the 3′ end of another nucleotide being covalently linked.These oligonucleotides are at least 14 nucleotides in length, but arepreferably 15 to 30 nucleotides long, with 16 to 29mers being the mostcommon.

These oligonucleotides can be prepared by the art recognized methodssuch as phosphoramidate or H-phosphonate chemistry which can be carriedout manually or by an automated synthesizer as described in Uhlmann etal. (Chem. Rev. (1990) 90:534-583) and Agrawal (Trends Biotechnol.(1992) 10:152-158).

The oligonucleotides of the invention may also be modified in a numberof ways without compromising their ability to hybridize to VEGF mRNA.For example, the oligonucleotides may contain at least one or acombination of other than phosphodiester internucleotide linkagesbetween the 5′ end of one nucleotide and the 3′ end of anothernucleotide in which the 5′ nucleotide phosphodiester linkage has beenreplaced with any number of chemical groups. Examples of such chemicalgroups include alkylphosphonates, phosphorothioates,phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters.

For example, U.S. Pat. No. 5,149,797 describes traditional chimericoligonucleotides having a phosphorothioate core region interposedbetween methylphosphonate or phosphoramidate flanking regions. U.S.patent application Ser. No. 08/516,454, filed on Aug. 9, 1995 discloses“inverted” chimeric oligonucleotides comprising one or more nonionicoligonucleotide region (e.g. alkylphosphonate and/or phosphoramidateand/or phosphotriester internucleoside linkage) flanked by one or moreregion of oligonucleotide phosphorothioate. Various oligonucleotideswith modified internucleotide linkages can be prepared according toknown methods (see, e.g., Goodchild (1990) Bioconjugate Chem. 2:165-187;Agrawal et al., (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083;Uhlmann et al. (1990) Chem. Rev. 90:534-583; and Agrawal et al. (1992)Trends Biotechnol. 10:152-158.

The phosphorothioate linkages may be mixed Rp and Sp enantiomers, orthey may be stereoregular or substantially stereoregular in either Rp orSp form (see Iyer et al. (1995) Tetrohedron Asymmetry 6:1051-1054).Oligonucleotides with phosphorothioate linkages can be prepared usingmethods well known in the field such as phosphoramidite (see, e.g.,Agrawal et al. (1988) Proc. Natl. Acad Sci. (USA) 85:7079-7083). or byH-phosphonate (see, e.g., Froehler (1986) Tetrahedron Lett.27:5575-5578) chemistry. The synthetic methods described in Bergot etal. (J. Chromatog. (1992) 559:35-42) can also be used.

Oligonucleotides which are self-stabilized are also considered to bemodified oligonucleotides useful in the methods of the invention (Tanget al. (1993) Nucleic Acids Res. 20:2729-2735). These oligonucleotidescomprise two regions: a target hybridizing region; and aself-complementary region having an oligonucleotide sequencecomplementary to a nucleic acid sequence that is within theself-stabilized oligonucleotide.

Other modifications include those which are internal or at the end(s) ofthe oligonucleotide molecule and include additions to the molecule ofthe internucleoside phosphate linkages, such as cholesterol,cholesterol, or diamine compounds with varying numbers of carbonresidues between the amino groups and terminal ribose, deoxyribose andphosphate modifications which cleave, or crosslink to the oppositechains or to associated enzymes or other proteins which bind to thegenome. Examples of such modified oligonucleotides includeoligonucleotides with a modified base and/or sugar such as arabinoseinstead of ribose, or a 3′, 5′-substituted oligonucleotide having asugar which, at both its 3′ and 5′ positions is attached to a chemicalgroup other than a hydroxyl group (at its 3′ position) and other than aphosphate group (at its 5′ position).

Other examples of modifications to sugars include modifications to the2′ position of the ribose moiety which include but are not limited to2′-O-substituted with an —O— lower alkyl group containing 1-6 saturatedor unsaturated carbon atoms, or with an —O-aryl, or allyl group having2-6 carbon atoms wherein such —O-alkyl, aryl or allyl group may beunsubstituted or may be substituted, (e.g., with halo, hydroxy,trifluoromethyl cyano, nitro acyl acyloxy, alkoxy, carboxy, carbalkoxyl,or amino groups), or with an amino, or halo group. None of thesesubstitutions are intended to exclude the native 2′-hydroxyl group inthe case of ribose or 2′-H— in the case of deoxyribose. PCT PublicationNo. WO 94/02498 discloses traditional hybrid oligonucleotides havingregions of 2′-O-substituted ribonucleotides flanking a DNA core region.U.S. patent application Ser. No. (47508-559), filed Aug. 9, 1995,discloses an “inverted” hybrid oligonucleotide which includes anoligonucleotide comprising a 2′-O-substituted (or 2′ OH, unsubstituted)RNA region which is in between two oligodeoxyribonucleotide regions, astructure that “inverted relative to the “traditional” hybridoligonucleotides. Nonlimiting examples of particularly usefuloligonucleotides of the invention have 2′-O-alkylated ribonucleotides attheir 3′, 5′, or 3′ and 5′ termini, with at least four or fivecontiguous nucleotides being so modified. Non-limiting examples of2′-O-alkylated groups include 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, and2′-O-butyls.

Other modified oligonucleotides are capped with a nucleaseresistance-conferring bulky substituent at their 3′ and/or 5′ end(s), orhave a substitution in one nonbridging oxygen per nucleotide. Suchmodifications can be at some or all of the internucleoside linkages, aswell as at either or both ends of the oligonucleotide and/or in theinterior of the molecule.

A nonlimiting list of useful oligonucleotides of the invention arelisted below in Table 1.

TABLE 1 TARGETED SEQ ID OLIGO SITE SEQUENCE (5′→3′) NO: H-3Ia1 81-62GCACCCAAGACAGCAGAAAG 2 H-3Ia2 81-62 GCACCCAAGACAGCAGAAAG 2 H-3Ia3 81-62GCACCCAAGACAGCAGAAAG 2 H-3Ia4 81-62 GCACCCAAGACAGCAGAAAG 2 H-3Ia5 81-62GCACCCAAGACAGCAGAAAG 2 H-3Ia6 81-62 GCACCCAAGACAGCAGAAAG 2 H-3Ia7 81-62GCACCCAAGACAGCAGAAAG 2 H-3Ia8 81-62 GCACCCAAGACAGCAGAAAG 2 H-3Ia9 81-62GCACCCAAGACAGCAGAAAG 2 H-3Ia10 81-62 GCACCCAAGACAGCAGAAAG 2 H-3I1 82-62TGCACCCAAGACAGCAGAAAG 3 H-3I2 82-62 TGCACCCAAGACAGCAGAAAG 3 H-3I3 82-62TGCACCCAAGACAGCAGAAAG 3 H-3I4 82-62 TGCACCCAAGACAGCAGAAAG 3 H-3I5 82-62TGCACCCAAGACAGCAGAAAG 3 H-3I6 82-62 TGCACCCAAGACAGCAGAAAG 3 H-3I7 82-62TGCACCCAAGACAGCAGAAAG 3 H-3I8 82-62 TGCACCCAAGACAGCAGAAAG 3 H-3I9 82-62TGCACCCAAGACAGCAGAAAG 3 H-3I10 82-62 TGCACCCAAGACAGCAGAAAG 3 H-3Ja183-62 ATGCACCCAAGACAGCAGAAAG 4 H-3Ja2 83-62 ATGCACCCAAGACAGCAGAAAG 4H-3Ja3 83-62 ATGCACCCAAGACAGCAGAAAG 4 H-3Ja4 83-62ATGCACCCAAGACAGCAGAAAG 4 H-3Ja5 83-62 ATGCACCCAAGACAGCAGAAAG 4 H-3Ja683-62 ATGCACCCAAGACAGCAGAAAG 4 H-3Ja7 83-62 ATGCACCCAAGACAGCAGAAAG 4H-3Ja8 83-62 ATGCACCCAAGACAGCAGAAAG 4 H-3Ja9 83-62ATGCACCCAAGACAGCAGAAAG 4 H-3Ja10 83-62 ATGCACCCAAGACAGCAGAAAG 4 H-3J184-62 AATGCACCCAAGACAGCAGAAAG 5 H-3J2 84-62 AATGCACCCAAGACAGCAGAAAG 5H-3J3 84-62 AATGCACCCAAGACAGCAGAAAG 5 H-3J4 84-62AATGCACCCAAGACAGCAGAAAG 5 H-3J5 84-62 AATGCACCCAAGACAGCAGAAAG 5 H-3J684-62 AATGCACCCAAGACAGCAGAAAG 5 H-3J7 84-62 AATGCACCCAAGACAGCAGAAAG 5H-3J8 84-62 AATGCACCCAAGACAGCAGAAAG 5 H-3J9 84-62AATGCACCCAAGACAGCAGAAAG 5 H-3J10 84-62 AATGCACCCAAGACAGCAGAAAG 5 H-3Xa185-62 CAATGCACCCAAGACAGCAGAAAG 6 H-3Xa2 85-62 CAATGCACCCAAGACAGCAGAAAG 6H-3Xa3 85-62 CAATGCACCCAAGACAGCAGAAAG 6 H-3Xa4 85-62CAATGCACCCAAGACAGCAGAAAG 6 H-3Xa5 85-62 CAATGCACCCAAGACAGCAGAAAG 6H-3Xa6 85-62 CAATGCACCCAAGACAGCAGAAAG 6 H-3Xa7 85-62CAATGCACCCAAGACAGCAGAAAG 6 H-3Xa8 85-62 CAATGCACCCAAGACAGCAGAAAG 6H-3Xa9 85-62 CAATGCACCCAAGACAGCAGAAAG 6 H-3Xa10 85-62CAATGCACCCAAGACAGCAGAAAG 6 H-3X1 86-62 CCAATGCACCCAAGACAGCAGAAAG 7 H-3X286-62 CCAATGCACCCAAGACAGCAGAAAG 7 H-3X3 86-62 CCAATGCACCCAAGACAGCAGAAAG7 H-3X4 86-62 CCAATGCACCCAAGACAGCAGAAAG 7 H-3X5 86-62CCAATGCACCCAAGACAGCAGAAAG 7 H-3X6 86-62 CCAATGCACCCAAGACAGCAGAAAG 7H-3X7 86-62 CCAATGCACCCAAGACAGCAGAAAG 7 H-3X8 86-62CCAATGCACCCAAGACAGCAGAAAG 7 H-3X9 86-62 CCAATGCACCCAAGACAGCAGAAAG 7H-3X10 86-62 CCAATGCACCCAAGACAGCAGAAAG 7 H-3Ya1 87-62TCCAATGCACCCAAGACAGCAGAAAG 8 H-3Ya2 87-62 TCCAATGCACCCAAGACAGCAGAAAG 8H-3Ya3 87-62 TCCAATGCACCCAAGACAGCAGAAAG 8 H-3Ya4 87-62TCCAATGCACCCAAGACAGCAGAAAG 8 H-3Ya5 87-62 TCCAATGCACCCAAGACAGCAGAAAG 8H-3Ya6 87-62 TCCAATGCACCCAAGACAGCAGAAAG 8 H-3Ya7 87-62TCCAATGCACCCAAGACAGCAGAAAG 8 H-3Ya8 87-62 TCCAATGCACCCAAGACAGCAGAAAG 8H-3Ya9 87-62 TCCAATGCACCCAAGACAGCAGAAAG 8 H-3Ya10 87-62TCCAATGCACCCAAGACAGCAG AAAG 8 H-Y1 88-62 CTCCAATGCACCCAAGACAGCAGAAAG 9H-Y2 88-62 CTCCAATGCACCCAAGACAGCAGAAAG 9 H-Y3 88-62CTCCAATGCACCCAAGACAGCAGAAAG 9 H-Y4 88-62 CTCCAATGCACCCAAGACAGCAGAAAG 9H-Y5 88-62 CTCCAATGCACCCAAGACAGCAGAAAG 9 H-Y6 88-62CTCCAATGCACCCAAGACAGCAGAAAG 9 H-Y7 88-62 CTCCAATGCACCCAAGACAGCAGAAAG 9H-Y8 88-62 CTCCAATGCACCCAAGACAGCAGAAAG 9 H-Y9 88-62CTCCAATGCACCCAAGACAGCAGAAAG 9 H-Y10 88-62 CTCCAATGCACCCAAGACAGCAGAAAG 9H-Za1 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 10 H-Za2 89-62GCTCCAATGCACCCAAGACAGCAGAAAG 10 H-Za3 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG10 H-Za4 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 10 H-Za5 89-62GCTCCAATGCACCCAAGACAGCAGAAAG 10 H-Za6 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG10 H-Za7 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 10 H-Za8 89-62GCTCCAATGCACCCAAGACAGCAGAAAG 10 H-Za9 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 10 H-Za10 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 10 H-Z1 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z2 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z3 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z4 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z5 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z6 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z7 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z8 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z9 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-Z10 90-62GGCTCCAATGCACCCAAGACAGCAGAAAG 11 H-3D1 80-63 CACCCAAGACAGCAGAAA 12 H-3D280-63 CACCCAAGACAGCAGAAA 12 H-3D3 80-63 CACCCAAGACAGCAGAAA 12 H-3D480-63 CACCCAAGACAGCAGAAA 12 H-3D5 80-63 CACCCAAGACAGCAGAAA 12 H-3D680-63 CACCCAAGACAGCAGAAA 12 H-3D7 80-63 CACCCAAGACAGCAGAAA 12 H-3D880-63 CACCCAAGACAGCAGAAA 12 H-3D9 80-63 CACCCAAGACAGC AGAAA 12 H-3D1080-63 CACCCAAGACAGCAGAAA 12 H-3E1 80-64 CACCCAAGACAGCAGAA 13 H-3E2 80-64CACCCAAGACAGCAGAA 13 H-3E3 80-64 CACCCAAGACAGCAGAA 13 H-3E4 80-64CACCCAAGACAGCAGAA 13 H-3E5 80-64 CACCCAAGACAGCAGAA 13 H-3E6 80-64CACCCAAGACAGCAGAA 13 H-3E7 80-64 CACCCAAGACAGCAGAA 13 H-3E8 80-64CACCCAAGACAGCAGAA 13 H-3E9 80-64 CACCCAAGACAGCAGAA 13 H-3E10 80-64CACCCAAGACAGCAGAA 13 H-3F1 80-65 CACCCAAGACAGCAGA 14 H-3F2 80-65CACCCAAGACAGCAGA 14 H-3F3 80-65 CACCCAAGACAGCAGA 14 H-3F4 80-65CACCCAAGACA GCAGA 14 H-3F5 80-65 CACCCAAGACAGCAGA 14 H-3F6 80-65CACCCAAGACAGCAGA 14 H-3F7 80-65 CACCCAAGACAGCAGA 14 H-3F8 80-65CACCCAAGACAGCAGA 14 H-3F9 80-65 CACCCAAGACA GCAGA 14 H-3F10 80-65CACCCAAGACAGCAGA 14 H-3G1 80-60 CACCCAAGACAGCAGAAAGTT 15 H-3G2 80-60CACCCAAGACAGCAGAAAGTT 15 H-3G3 80-60 CACCCAAGACAGCAGAAAGTT 15 H-3G480-60 CACCCAAGACAGCAGAAAGTT 15 H-3G5 80-60 CACCCAAGACAGCAGAAAGTT 15H-3G6 80-60 CACCCAAGACAGCAGAAAGTT 15 H-3G7 80-60 CACCCAAGACAGCAGAAAGTT15 H-3G8 80-60 CACCCAAGACAGCAGAAAGTT 15 H-3G9 80-60CACCCAAGACAGCAGAAAGTT 15 H-3G10 80-60 CACCCAAGACAGCAGAAAGTT 15 H-3H180-58 CACCCAAGACAGCAGAAAGTTCAT 16 H-3H2 80-58 CACCCAAGACAGCAGAAAGTTCAT16 H-3H3 80-58 CACCCAAGACAGCAGAAAGTTCAT 16 H-3H4 80-58CACCCAAGACAGCAGAAAGTTCAT 16 H-3H5 80-58 CACCCAAGACAGCAGAAAGTTCAT 16H-3H6 80-58 CACCCAAGACAGCAGAAAGTTCAT 16 H-3H7 80-58CACCCAAGACAGCAGAAAGTTCAT 16 H-3H8 80-58 CACCCAAGACAGCAGAAAGTTCAT 16H-3H9 80-58 CACCCAAGACAGCAGAAAGTTCAT 16 H-3H10 80-58CACCCAAGACAGCAGAAAGTTCAT 16

Preferably, the nucleotides bolded in the oligonucleotides above are2′-O-alkylated, and all of the nucleotides are linked vianon-phosphodiester internucleotide linkages such as phosphorothioates.

The preparation of these modified oligonucleotides is well known in theart (reviewed in Agrawal (1992) Trends Biotechnol. 10:152-158; Agrawalet al. (1995) Curr. Opin. Biotechnol. 6:12-19). For example, nucleotidescan be covalently linked using art-recognized techniques such asphosphoramidate, H-phosphonate chemistry, or methylphosphoramidatechemistry (see, e.g., Uhlmann et al. (1990) Chem. Rev. 90:543-584;Agrawal et al. (1987) Tetrahedron Lett. 28: (31) :3539-3542); Carutherset al. (1987) Meth. Enzymol. 154:287-313; U.S. Pat. No. 5,149,798).Oligomeric phosphorothioate analogs can be prepared using methods wellknown in the field such as methoxyphosphoramidite (see, e.g., Agrawal etal. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083) or H-phosphonate(see, e.g., Froehler (1986) Tetrahedron Lett. 27:5575-5578) chemistry.The synthetic methods described in Bergot et al. (J. Chromatog. (1992)559:35-42) can also be used.

The synthetic antisense oligonucleotides of the invention in the form ofa therapeutic formulation are useful in treating diseases, anddisorders, and conditions associated with angiogenesis andneovascularization including, but not limited to, retinalneovascularization, tumor growth, wound healing, bullous pemphigoid,dermatitis herpetiformis, erythema multiforme, and psoriasis. In suchmethods, a therapeutic amount of a synthetic oligonucleotide of theinvention and effective in inhibiting the expression of vascularendothelial growth factor is administered to a cell. This cell may bepart of a cell culture, a neovascularized tissue culture, or may be partor the whole body of an animal such as a human or other mammal.Administration may be topical, intralesional, bolus, intermittent, orcontinuous, depending on the condition and response, as determined bythose with skill in the art. In some preferred embodiments of themethods of the invention described above, the oligonucleotide isadministered locally (e.g., intraocularly or interlesionally) and/orsystemically. The term “local administration” refers to delivery to adefined area or region of the body, while the term “systemicadministration is meant to encompass delivery to the whole organism byoral ingestion, or by intramuscular, intravenous, subcutaneous, orintraperitoneal injection.

Such methods can be used to treat retinopathy of prematurity (ROP),diabetic retinopathy, age-related macular degeneration, sickle cellretinopathy, neovascular glaucoma, retinal vein occlusion, and otherhypoxic diseases.

The synthetic oligonucleotides of the invention may be used as part of apharmaceutical composition when combined with a physiologically and/orpharmaceutically acceptable carrier. The characteristics of the carrierwill depend on the route of administration. Such a composition maycontain, in addition to the synthetic oligonucleotide and carrier,diluents, fillers, salts, buffers, stabilizers, solubilizers, and othermaterials well known in the art. The pharmaceutical composition of theinvention may also contain other active factors and/or agents whichenhance inhibition of VEGF expression or which will reduceneovascularization. For example, combinations of syntheticoligonucleotides, each of which is directed to different regions of theVEGF mRNA, may be used in the pharmaceutical compositions of theinvention. The pharmaceutical composition of the invention may furthercontain nucleotide analogs such as azidothymidine, dideoxycytidine,dideosyinosine, and the like. Such additional factors and/or agents maybe included in the pharmaceutical composition to produce a synergisticeffect with the synthetic oligonucleotide of the invention, or tominimize side-effects caused by the synthetic oligonucleotide of theinvention. Conversely, the synthetic oligonucleotide of the inventionmay be included in formulations of a particular anti-VEGF oranti-neovascularization factor and/or agent to minimize side effects ofthe anti-VBGF factor and/or agent.

The pharmaceutical composition of the invention may be in the form of aliposome in which the synthetic oligonucleotides of the invention iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layerswhich are in aqueous solution. Suitable lipids for liposomal formulationinclude, without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like. Oneparticularly useful lipid carrier is lipofectin. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No.4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323. Thepharmaceutical composition of the invention may further includecompounds such as cyclodextrins and the like which enhance delivery ofoligonucleotides into cells, as described by Zhao et al. (in press), orslow release polymers.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,healing of chronic conditions characterized by neovascularization or areduction in neovascularization, itself, or in an increase in rate ofhealing of such conditions, or in a reduction in aberrant epidermallesions. When applied to an individual active ingredient, administeredalone, the term refers to that ingredient alone. When applied to acombination, the term refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of one, two, or more of the syntheticoligonucleotides of the invention is administered to a subject afflictedwith a disease or disorder related to neovascularization, or to a tissuewhich has been neovascularized. The synthetic oligonucleotide of theinvention may be administered in accordance with the method of theinvention either alone of in combination with other known therapies forneovascularization, angiogenesis, or dermatoses related thereto. Whenco-administered with one or more other therapies, the syntheticoligonucleotide of the invention may be administered eithersimultaneously with the other treatment(s), or sequentially. Ifadministered sequentially, the attending physician will decide on theappropriate sequence of administering the synthetic oligonucleotide ofthe invention in combination with the other therapy.

Administration of the synthetic oligonucleotide of the invention used inthe pharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such astopical or intralesional administration, intraocular, oral ingestion,inhalation, or cutaneous, subcutaneous, intramuscular, or intravenousinjection.

When a therapeutically effective amount of synthetic oligonucleotide ofthe invention is administered orally, the synthetic oligonucleotide willbe in the form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%synthetic oligonucleotide and preferably from about 25 to 90% syntheticoligonucleotide. When administered in liquid form, a liquid carrier suchas water, petroleum, oils of animal or plant origin such as peanut oil,mineral oil, soybean oil, sesame oil, or synthetic oils may be added.The liquid form of the pharmaceutical composition may further containphysiological saline solution, dextrose or other saccharide solution, orglycols such as ethylene glycol, propylene glycol or polyethyleneglycol. When administered in liquid form, the pharmaceutical compositioncontains from about 0.5 to 90% by weight of the syntheticoligonucleotide and preferably from about 1 to 50% syntheticoligonucleotide.

When a therapeutically effective amount of synthetic oligonucleotide ofthe invention is administered by intravenous, subcutaneous,intramuscular, intraocular, or intraperitoneal injection, the syntheticoligonucleotide will be in the form of a pyrogen-free, parenterallyacceptable aqueous solution. The preparation of such parenterallyacceptable solutions, having due regard to pH, isotonicity, stability,and the like, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, subcutaneous, intramuscular,intraperitoneal, or intraocular injection should contain, in addition tothe synthetic oligonucleotide, an isotonic vehicle such as SodiumChloride Injection, Ringer's Injection, Dextrose Injection, Dextrose andSodium Chloride Injection, Lactated Ringer's Injection, or other vehicleas known in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

When administered topically or intralesionally as a liquid, dosesranging from 0.01% to 10% (weight/volume) may be used. When administeredin liquid form, a liquid carrier such as water, petroleum, oils ofanimal or plant origin such as peanut oil, mineral oil, soybean oil,sesame oil, or synthetic oils may be added. Topical administration maybe by liposome or transdermal time-release patch.

The amount of synthetic oligonucleotide in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patent has undergone. Ultimately, the attendingphysician will decide the amount of synthetic oligonucleotide with whichto treat each individual patient. Initially, the attending physicianwill administer low doses of the synthetic oligonucleotide and observethe patient's response. Larger doses of synthetic oligonucleotide may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. It iscontemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 10 μgto about 20 mg of synthetic oligonucleotide per kg body or organ weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. Ultimately the attending physicianwill decide on the appropriate duration of intravenous therapy using thepharmaceutical composition of the present invention.

Some diseases lend themselves to acute treatment while others require tolonger term therapy. Proliferative retinopathy can reach a threshold ina matter of days as seen. in ROP, some cases of diabetic retinopathy,and neovascular glaucoma. Premature infants are at risk forneovascularization around what would be 35 weeks gestation, a few weeksafter birth, and will remain at risk for a short period of time untilthe retina becomes vascularized. Diabetic retinopathy can be acute butmay also smolder in the proliferative phase for considerably longer.Diabetic retinopathy will eventually become quiescent as thevasoproliferative signal diminishes with neovascularization ordestruction of the retina.

Both acute and long term intervention in retinal disease are worthygoals. Intravitreal injections of oligonucleotides against VEGF can bean effective means of inhibiting retinal neovascularization in an acutesituation. However for long term therapy over a period of years,systemic delivery (intraperitoneal, intramuscular, subcutaneous,intravenous) either with carriers such as saline, slow release polymers,or liposomes should be considered.

In some cases of chronic neovascular disease, systemic administration ofoligonucleotides may be preferable. Since the disease process concernsvessels which are abnormal and leaky, the problem of passage through theblood brain barrier may not be a problem. Therefore, systemic deliverymay prove efficacious. The frequency of injections is from continuousinfusion to once a month, depending on the disease process and thebiological half life of the oligonucleotides.

In addition to inhibiting neovascularization in vivo, antisenseoligonucleotides specific for VEGF are useful in determining the role ofthis cytokine in processes where neovascularization is involved. Forexample, this technology is useful in in vitro systems which mimic bloodvessel formation and permeability, and in in vivo system models ofneovascularization, such as the murine model described below.

A murine model of oxygen-induced retinal neovascularization has beenestablished which occurs in 100% of treated animals and is quantifiable(Smith et al. (1994) Invest. Ophthamol. Vis. Sci. 35:101-111). Usingthis model, a correlation has been determined between increasingexpression of VEGF message and the onset of retinal neovascularizationin the inner nuclear and ganglion cell layers (i.e., in Müller cells)(Pierce et al. (1995) Proc. Natl. Acad. Sci. (USA) (in press). Thisresult has been confirmed by Northern blot and in situ hybridizationanalysis of whole retinas at different time points during thedevelopment of neovascularization (Pierce et al., ibid.).

Oligonucleotides of the invention are also useful in a method ofreducing the expression of VEGF. The target VEGF expression can be invitro or in any cell which expresses VEGF. In this method, nucleic acidspecific for VEGF is contacted with an oligonucleotide of the inventionsuch that transcription of the nucleic acid to mRNA and/or protein isreduced or inhibited.

That oligonucleotides of the invention can inhibit VEGF expression atthe protein level can be demonstrated using an ELISA which specificallydetects human VEGF and a VEGF-expressing cell line such as a humanglioblastoma (e.g., U373 ATCC Ac. no. HTB17, American Type CultureCollection, Rockville, Md.) or a human melanoma (e.g., SK-MEL-2, ATCCAc. no. HTB68, American Type Culture Collection, Rockville, Md.; orM21). Briefly, when a human glioblastoma cell line U373 and a humanmelanoma cell line E21 were treated with VEGF-specific oligonucleotidesof the invention, these cells stop expressing VEGF in asequence-specific manner, as shown in FIGS. 2, 3, and 4, and in FIG. 5,respectively. FIG. 6 demonstrates that modification of theoligonucleotides does not reduce their inhibitory activity.Oligonucleotides of the invention also reduced VEGF mRNA expression, asdemonstrated by the Northern analyses described in EXAMPLE 4 below.

VEGF's role in tumor formation in vivo can be demonstrated using anathymic mouse injected with as an animal model. M21 cells are known togenerate palpable tumors in mice in about 1 to 1.5 weeks. Alternately, aU373 cell line which has been passed through an athymic mouse in thepresence of Engelbreth Holm Swarm (EMS) tumor matrix (Matrigel™,Collaborative Research, Waltham, Mass.) may be used. When mice areinjected with VEGF-specific oligonucleotides of the invention, therewill be a reduction in tumor weight and volume if VEGF expression isreduced by oligonucleotides or pharmaceutical formulations of theinvention.

That VEGF plays a role in retinal neovascularization has been shownusing the murine model of neovascularization described above. Threeindependent experiments were performed using antisense oligonucleotidesspecific for VEGF (JG-3 (SEQ ID NO:17), JG-4, (SEQ ID NO:18), and Vm(SEQ ID NO:19), and a corresponding sense oligonucleotide (V2 (SEQ IDNO:20). These oligonucleotides were designed using the known nucleicsequence of murine VEGF (Claffee et al. (1992) J. Biol. Chem.267:16317-16322). The sequence of the Vm oligonucleotide is targeted tothe sequence surrounding the translational TGA stop site (TGA). Thesequence of JG-4 is targeted to the sequence 5′ to and containing theATG of the translational start site of the murine VEGF molecule. Thesequence of JG-3 is targeted to the 5′ untranslated region, and the V2sense sequence is targeted to the sequence surrounding the translationalstart site (ATG).

That oligonucleotides of the invention can down regulate the expressionVEGF in human epidermal keratinocytes, and hence, have a clinicalapplication in the treatment of psoriasis and the bullous diseases, weredemonstrated in culture system that mimics the in vivo psoriasicconditions of human skin cells. This system was developed with theknowledge that TGFα, known to be overexpressed in the epidermis ofpsoriasic skin lesions, induces VEGF expression at the RNA and proteinlevel in cultured human keratinocytes (Detmar et al. (1995) J. Invest.Dermatol. 105:44-50). Thus, in the test system, normal human epidermalkeratinocytes were cultured in the presence of TGFα.Oligodeoxynucleotides of the invention used in this study were targetedto a region adjacent to the translational start site which is conservedin all VEGF family members. These oligonucleotides can be taken up bypost-confluent NAEK's as determined by flow cytometric studies whichshowed an increase in fluorescence of cells treated withfluorescein-labelled oligonucleotides. These oligonucleotides, whichinhibit CoCl₂ and hypoxia induced VBGF/VPF expression at the levels ofmRNA and protein, were also found to inhibit the TGFα-dependentinduction of VEGF expression in normal human epidermal keratinocytes(FIG. 7).

In order to see if a decrease in VEGF/VPF protein was paralleled by adecrease in VEGF mRNA, confluent keratinocytes were treated with orwithout phosphorothioate oligonucleotides and lipofectin for eight hoursfollowed by replacement of the medium with basal medium containingelevated levels of TGFα or the medium, alone. Supernatants werecollected at 24 and 48 hours and analyzed by Northern blot for RNA andby ELISA for protein expression. Total RNA was extracted from the cellsas described in the exemplification, below, and analyzed by Northernblot. The results in FIG. 8 show an increase of VEGF RNA at 24 and 48hours in cells treated with TGFα in the presence or absence of sense ormismatched phosphorothioate oligonucleotides. Cells treated with theantisense oligonucleotide phosphorothioates showed a reduced level ofVEGF RNA to near basal levels for both time points examined. Thisdecrease in VEGF RNA levels by antisense oligonucleotidephosphorothioate treatment was paralleled by a decrease in VEGF proteinaccumulation in the media as assayed by capture ELISA. Analysis of thecell culture media for control and mismatch oligonucleotidephosphorothioate-treated cells showed little or no effect on theproduction of VEGF protein levels.

To determine how long inhibition of VEGF protein expression ismaintained after treatment with antisense oligonucleotides of theinvention, cells were treated with oligonucleotide phosphorothioates andlipofectin for eight hours. Then medium containing TGF4 was added.Supernatants were collected at 48, 72, 96, and 120 hourspost-oligonucleotide phosphorothioate treatment and TGFα addition, andassayed by capture ELISA for VEGF protein. The results shown in FIG. 9demonstrate that inhibition of VEGF protein expression is maintained for48 hours and continues out to 120 hours. The control senseoligonucleotide phosphorothioate, H3S (SEQ ID NO:21), showed only aslight stimulatory effect. All keratinocytes appeared viable by lightmicroscopy.

The following examples illustrate the preferred modes of making andpracticing the present invention, but are not meant to limit the scopeof the invention since alternative methods may be utilized to obtainsimilar results.

EXAMPLE 1 Preparation of VEGF-Specific Oligonucleotides

Human VEGF cDNA is transcribed in vitro using an in vitro eucaryotictranscription kit (Stratagene, La Jolla, Calif.). The RNA is labelledwith ³²P using T-4 polynucleotide kinase as described by (Sambrook etal. (1989) Molecular Cloning; a Laboratory Manual, Cold Spring HarborLaboratory Press, NY, Vol. 1, pp. 5.71). The labelled RNA is incubatedin the presence of a randomer 20mer library and RNase H, an enzyme whichcleaves RNA-DNA duplexes (Boehringer Mannheim, Indianapolis, Ind.).Cleavage patterns are analyzed on a 6% polyacrylamide urea gel. Thespecific location of the cleaved fragments is determined using a humanVEGF sequence ladder (Sequenase Kit, United States Biochemical,Cleveland, Ohio).

Oligonucleotides having sequences complementary to VEGF nucleic aciddetermined as described above were synthesized on a Pharmacia GeneAssembler series synthesizer using the phosphoramidite procedure (see,e.g., Uhlmann et al. (Chem. Rev. (1990) 90:534-583; Agrawal (1992)Trends in Biotech. 10:152-158; Agrawal et al. (1995) Curr. Opin.Biotechnol. 6:12-19). Following assembly and deprotection,oligonucleotides were ethanol precipitated twice, dried, and suspendedin phosphate-buffered saline (PBS) at the desired concentration.

The purity of these oligonucleotides was tested by capillary gelelectrophoreses and ion exchange HPLC. Endotoxin levels in theoligonucleotide preparation was determined using the Luminous AmebocyteAssay (Bang (1953) Biol. Bull. (Woods Hole, Mass.) 105:361-362).

EXAMPLE 2 Human Cell Culture

U373 human glioblastoma cells (American Type Culture Collection,Rockville, Md., ATCC Ac. no. HTB17) were cultured in Dulbecco's modifiedEarls (DME) medium containing glucose (4500 mg/ml) and 2 mM glutamate(Mediatech, Washington, D.C.) supplemented with penicillin/streptomycin(100 IU/MI/100 mcg/ml, Mediatech, Washington, D.C.). The cells werecultured at 37° C. under 10% CO₂. The cells were plated in 96 welltissue culture dishes (Costar Corp., Cambridge, Mass.) and maintained asabove. The cells were placed under anoxic conditions for 18-20 hoursusing an anaerobic chamber (BBL Gas Pak, Cockeysville, Md.) or in thepresence of 250 μM CoCl².

Normal human epidermal keratinocytes (Clonetics, San Diego, Calif.) weregrown as recommended in Keratinocyte Growth Medium, KGM (Clonetics, SanDiego, Calif.) or Keratinocyte Basal Medium, KBM (Clonetics, San Diego,Calif.). Cells for all experiments were used at passage 2 or 3 in 10%CO₂. For antisense experiments, cells were treated post confluence in P100's or 24 well plates (Costar®, Cambridge, Mass.) with Lipofectin™(Gibco BRL, Gaithersburg, Md.) and phosphorothioateoligodeoxynucleotides in KBM for 6-8 hours at which time the media wasreplaced with KBM with or without TGFα (Gibco BRL, Gaithersburg, Md.) at100 ng/ml. The medium and cells were collected at 24 or 48 hourspost-TGFα induction and used for ELISA and RNA analysis.

EXAMPLE 3 ELISA VEGF Protein Study

U373 glioblastoma cells were plated in a 96 well tissue culture dish andtreated overnight with varying concentrations of antisenseoligonucleotides against human VEGF in the presence of 5 μg/mllipofectin. The cells were refed after 7 to 12 hours with fresh mediaand allowed to recover for 5 to 7 hours. The dishes were placed underhypoxic conditions for 18 to 20 hours using an anaerobic chamber (GasPac, Cockeysville, Md.) or in the presence of 250 μM CoCl₂. Cellsmaintained under normoxic conditions served as uninduced controls. Themedia was analyzed using the antigen capture ELISA assay described below(approximately 24 hours post treatment).

The culture medium from the cells described in EXAMPLE 2 was analyzedfor VEGF protein as follows. 96-well plates (Maxizorb ELISA Nunc A/S,Camstrup, Denmark) were treated overnight at 4° C. with 100 μl/well ofthe capture antibody, a monoclonal antibody against human VEGF (R&DSystems, Minneapolis, Minn., 2.5 μg/ml in 1×PBS). The wells were washedthree times with 1×PBS/0.05% Tween-20 (United States Biochemical,Cleveland, Ohio) using a plate washer (Dynatech, Gurnsey ChannelIslands). Non-specific binding sites in the wells were blocked by adding2% normal human serum (200 μl) and incubating the plate at 37° C. for 2hours. This blocking solution was removed and 200 μl conditioned mediumcontaining human VEGF added to each well and incubated at 37° C. for 2to 3 hours or overnight at 4° C. The plates were washed as describedabove. 100 μl of the primary antibody (618/619, 2 μg/ml in normal humanserum) was added to each well and incubated at 37° C. for 1 to 2 hours.The primary antibody was an affinity purified rabbit anti-human VEGFpolyclonal). The plates were washed as described above. 100 μl of thedetection antibody, a horse radish peroxidase-labelled goat anti-rabbitIgG monoclonal antibody (1:10,000, Vector Laboratories, Burlinggame,Calif.), was added to each well and incubated at 37° C. for 1 hour. Theplates were washed as described above. The wells were developed usingthe TMB microwell peroxidase developing system (Kirkegaard and Perry,Gaithersburg, Md.), and quantified at 450 nm using a Ceres 900 platereader (Bio-Tek Instruments, Inc., Winooski, Vt.). The linear range ofthis assay is between 2 ng and 0.01 ng human VEGF. Representativeresults are shown in FIGS. 2-6.

EXAMPLE 4 Northern Blot Analysis

In order to determine the level at which inhibition of VEGF expressionoccurs in cells in the presence of an oligonucleotide of the invention,Northern blotting was carried out. Human U373 cells cultured asdescribed in EXAMPLE 2 above were plated in 100 mm tissue culture dishesand treated for 12 hours in the presence of 5 μg/ml lipofectin(Gibco-BRL, Gaithersburg, Md.) as a lipid-carrier with oligonucleotideH-3 (SEQ ID NO:1) and sense control (SEQ ID NO:21) at 0.05 μM, 0.5 μM,and 2.0 μM, respectively. The cells were refed after 7 to 8 hours withfresh media. The cells were placed in hypoxia for 18 to 20 hours or inthe presence of 250 μM CoCl², and total RNA was isolated using Trizol™(Gibco-BRL, Gaithersburg, Md.) and the single-step acid guanidiniumthiocyanate-phenol-chloroform extraction method described by Chomczynskiet al. (Anal. Biochem. (1987) 162:156-159). Northern blotting wasperformed according to the methods of Sambrook et al. (MolecularCloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, NY)(1989) Vol. 1, pp. 7.38) or Arcellana-Panlilio et al. (Meth. Enz. (1993)225:303-328). All RNA signals were quantified on a Phosphorimager(BioRad, Hercules, Calif.) and normalized using the 36B4 cDNA probe(Laborda (1991) Nucleic Acids Res. 19:3998). RNA expression was reducedin the presence of VEGF-specific oligonucleotides of the invention, andis not significantly affected by the presence of control senseoligonucleotide.

Alternatively, RNA was prepared from normal human epidermalkeratinocytes, as described in EXAMPLE 2, using TRIzol™ (Gibco BRL,Gaithersburg, Md.) as manufacturers directions. Ten micrograms of totalRNA was electrophoresed on a 1% formaldehyde gel and transferred ontoZeta-Probe® (BioRad, Hercules, Calif.). The blot was hybridized to arandomer primed simian VEGF cDNA and a cDNA, 36B4, a ribosomalassociated protein to control for loading. Washes were performed asmanufacturers directions. The blot was exposed to BIOMAX™MR Film(Eastman Kodak Company, Rochester, N.Y.).

Data from the Northern blot demonstrates a disease in the amount of VEGFRNA in tells treated with the antisense molecule H3 and little or noeffect with the control sense phosphorothioate oligonucleotide, H3S.

EXAMPLE 5 In vivo Studies

A. Matrigel Studies

U373 glioblastoma cells were treated with 0.5 μM antisensephosphorothioate oligodeoxynucleotide (H-3, SEQ ID NO:1) or control(H3-sense phosphorothioate oligonucleotide; SEQ ID NO:21) for 7 hours inthe presence of 5 μg/μl Lipofectin (Gibco-BRL, Gaithersburg, Md.). 1×10⁶oligonucleotide-treated cells were mixed with 250 μl Matrigel™(Collaborative Research, Waltham, Mass.; 10-12 mg/ml) and injectedsubcutaneously into 6-8 week old athymic mice (about 20 g) (CharlesRiver Laboratories, Wilmington, Mass.) on both the left and right sides.These cells respond to hypoxia and express increased levels of VEGF. Themice were maintained ad libitum and sacrificed 8 days post injection.The skin was dissected to expose the Matrigel pellet. Gross photographyof the surrounding blood vessels was performed with a Zeiss Macroscope.The Matrigel plugs were removed and fixed in formalin for paraffinembedding and histological analysis. Tissue sections were stained withhematoxylin and eosin for quantitation of blood vessel growth into theMatrigel plug.

The injection of Matrigel alone resulted in a clear plug with noapparent angiogenesis. Matrigel plugs combined with U373 glioblastomacells contained visible hemhorraging. In addition, the capillary bedsurrounding the plug was more dense and the blood vessels were moretortuous. Athymic mice injected with Matrigel plugs combined withantisense oligonucleotide-treated cells generated less angiogenesis thanthe mice injected with Matrigel plugs and either untreated cells orcells pretreated with the control oligonucleotide. Matrigel plugscontaining antisense treated cells also had less visible hemhorraging.The results suggest that antisense oligonucleotide treatment inhibitVEGF-induced angiogenesis.

B. Tumor Studies

6 week old athymic mice (about 20 g) are purchased from Charles RiverLaboratories. Human melanoma M21 cells or human glioblastoma U373 cellswhich have been passaged through athymic mice in the presence ofMatrigel are injected subcutaneously (2-20+10⁶) into the flank ofathymic mice. Palpable tumors are generated in 1-2 weeks. Subcutaneousantisense or sense control oligonucleotide injections begin one dayfollowing the injection of the tumor cells. The concentration ofoligonucleotide is determined and ranges between 5 and 50 mg/kg. Animalsare then injected over a period of three weeks. They are then sacrificedand, the tumors removed. Tumors are analyzed initially for weight andvolume. In addition, analysis includes sectioning and staining forVEGF/VPF protein using an anti-human VEGF/VPF monoclonal antibody (R&DSystems, Minneapolis, Minn.) or VEGF/VPF RNA using in situ hybridizationtechniques. Mice injected with antisense oligonucleotides of theinvention are expected to have smaller tumors than those injected withvehicle or the control.

EXAMPLE 6 Animal Model of Retinal Neovascularization

A. Preparation of Oligonucleotides

Synthesis of the following oligonucleotides:

JG-3 (SEQ ID NO:17), JG-4 (SEQ ID NO:18), Vm (SEQ ID NO:19), and V2 (SEQID NO:20), was performed as described in Example 1.

B. Preparation of Animal Model

Seven day postnatal mice (P7, C57b1/6J, (Children's Hospital BreedingFacilities, Boston, Mass.) were exposed to 5 days of hyperoxicconditions (75+/−2%) oxygen in a sealed incubator connected to a Bird3-M oxygen blender (flow rate: 1.5 liters/minute; Bird, Palm Springs,Calif.). The oxygen concentration was monitored by means of an oxygenanalyzer (Beckman, Model D2, Irvine, Calif.). After 5 days (P12), themice were returned to room air. Maximal retinal neovascularization wasobserved 5 days after return to room air (P17). After P21, the level ofretinal neovascularization was just beginning to regress.

C. Treatment

After mice had been removed from oxygen, antisense oligonucleotides wereinjected into the vitreous with a Hamilton syringe and a 33 gauge needle(Hamilton Company, Reno, Nev.). The animals were anesthetized for theprocedure with Avertin ip. The mice were given a single injection ofantisense oligonucleotides (or sense or non-sense controls) at P12achieving a final concentration of approximately 30-50 μM. The animalswere sacrificed at P17 with tribromoethanol ip (0.1 ml/g body weight)and cervical dislocation.

D. Microscopy

The eyes were enucleated, fixed in 4% paraformaldehyde, and embedded inparaffin. Serial sections of the whole eyes were cut sagittally, throughthe cornea, and parallel to the optic nerve. The sections were stainedwith hematoxylin and periodic acid-Schiff (PAS) stain. The extent ofneovascularization in the treated eyes was determined by countingendothelial cell nuclei extending past. the internal limiting membraneinto the vitreous. Nuclei from new vessels and vessel profiles could bedistinguished from other structures in the retina and counted incross-section with light microscopy. Additional eyes were sectioned andexamined by in situ hybridization to a VEGF probe.

To examine the retinal vasculature using fluorescein-dextran, the micewere perfused with a 50 mg/ml solution of high molecular weightfluorescein-dextran (Sigma Chemical Company, St. Louis, Mo.) in 4%paraformaldehyde. The eyes were enucleated, fixed in paraformaldehyde,and flat-mounted with glycerol-gelatin. The flat-mounted retinas wereviewed and photographed by fluorescence microscopy using an Olympus BX60fluorescence microscope (Olympus America Corp., Bellingham, Mass.).

EXAMPLE 7 Inhibition of VEGF Expression in Human Epidermal Keratinocytes

Normal human epidermal keratinocytes were purchased from CloneticsR (SanDiego, Calif.) as grown as recommended in Keratinocyte Growth Medium(KGM) or Keratinocyte Basal Medium (KBM) supplemented with TGF alpha(Gibco BRL, Gaithersburg, Md.). Cells for all experiments were used atpassages 2 or 3, and cultured in a humidified incubator with 10% CO2 at37° C. For antisense treatment, the medium from confluent cells wasaspirated and replaced with KBM containing lipofectin (Gibco BRL,Gaithersburg, Md.). Oligonucleotide phosphorothioates described below inTABLE 2 of the appropriate concentration were then added.

TABLE 2 SEQ.ID. Oligo Sequence (5′-3′) NO: H3 CACCCAAGACAGCAGAAAG 1 H3SCTTTCTGCTGTCTTGGGTG 21 5′1bpMM¹ CTCCCAAGACAGCAGAAAG 22 5′2bpMW²CTGCCAAGACAGCAGAAAG 23 cen4bpMM³ CACCCAACTCTCCAGAAAG 24 3′1bpMM⁴CACCCAAGACAGCAGAATG 25 3′2bpMM⁵ CACCCAAGACAGCAGATTG 26 ¹-5′ one basepair mismatch ²-5′ two base pair mismatch ³-central four base pairmismatch ⁴-3′ one base pair mismatch ⁵-3′ two base pair mismatch

All experiments were performed with 0.5 uM oligodeoxynucleotides, unlessotherwise stated. After eight hours, the medium was replaced with KBM inthe presence of TGF alpha. The medium was collected at 24 or 48 hourspost-TGF alpha addition and analyzed by ELISA as described below. TotalRNA was obtained from the cells and analyzed by Northern blot asdescribed below.

96 well plates (Nunc Maxisorp™, Nunc, Denmark) were coated overnight at4° C. with anti-human VEGF monoclonal antibody (R & D Systems,Minneapolis, Minn.), and blocked for 2 hours with 2% normal human serum.Cell supernatants were added and incubated overnight. Plates were washed3 times in 0.05% Tween 20 (USB, Cleveland, Ohio)/PBS (Cellgro®, Herndon,Vir.), on a plate washer (Dynatech, Chantilly, Vir.). An affinitypurified rabbit anti-human VEGF polyclonal antibody (1.5 μg/ml) wasadded and allowed to incubate for 2 hours at 37° C. Plates were washedas described above and a 1:10000 dilution of horse radish peroxidase(HRP)-conjugated goat-anti-rabbit IgG secondary antibody, (Kirkegaardand Perry, Gaithersburg, Md.) was added and allowed to incubate for 1hour. ELISA plates were developed using the TMB Microwell PeroxidaseSubstrate System (Kirkegaard and Perry, Gaithersburg, Md.) and read on aCeres 900 microplate reader (Bio-tek Instruments, Winooski, Vt.).

Data from the ELISA shows a specific inhibition of TGFα-induced VEGF tonear basal levels by oligonucleotide H3 (SEQ ID NO:1). The controloligonucleotide, H3S sense (SEQ ID NO:21), shows levels equal to cellstreated with TGFα alone. These results were confirmed at 24 and 48 hourtimepoints.

EXAMPLE 8 Flow Cytometry

A fluorescein moiety was attached to the 5′ end of the H3oligonucleotide phosphorothioate for flow cytometry experiments. Foruptake analysis, cells were grown in a 6 well plate in KGM. Two dayspost-confluence cells were treated in KBM with or withoutfluorescein-labelled oligonucleotide phosphorothioates in the presenceor absence of lipofectin for eight hours. Cells were trypsinized, washedtwice with PBS, and analyzed on a flow cytometer (Coulter Epics XL,Hialeah, Fla.) gating on live cells. Fluorescence was monitored at 488nm with a 525 nm band pass filter.

EXAMPLE 9 Northern Blotting

Total RNA was obtained from cells and analyzed by Northern blot asfollows. RNA was prepared using Trizol (Gibco BRL, Gaithersburg, Md.) asper manufacturers directions. Twenty micrograms of total RNA wasseparated on a 1% formaldehyde denaturing agarose gel (Chomczynski andMakey, (1994) Anal. Biochem 221:303-305) transferred to Zeta-Probe®(Biorad, Hercules, Calif.) nylon membrane, and hybridized to a randomprimer labeled simian VEGF/VPF cDNA probe. A probe for 36B4, a ribosomalassociated protein, was used to control for loading. The blot wasscanned using a Biorad GS525 Molecular Imager® system. Analysis of theacquired image was performed using the Molecular Analyst software. Thesignals were quantified by volume analysis (mm2*pixil density). Thenumbers shown are the ratios of the VEGF/VPF signal divided by theribosomal protein, 36B4, signal.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

26 19 base pairs nucleic acid single linear cDNA NO YES 1 CACCCAAGACAGCAGAAAG 19 20 base pairs nucleic acid single linear cDNA/RNA NO YES 2GCACCCAAGA CAGCAGAAAG 20 21 base pairs nucleic acid single linearcDNA/RNA NO YES 3 TGCACCCAAG ACAGCAGAAA G 21 22 base pairs nucleic acidsingle linear cDNA/RNA NO YES 4 ATGCACCCAA GACAGCAGAA AG 22 23 basepairs nucleic acid single linear cDNA/RNA NO YES 5 AATGCACCCA AGACAGCAGAAAG 23 24 base pairs nucleic acid single linear cDNA/RNA NO YES 6CAATGCACCC AAGACAGCAG AAAG 24 25 base pairs nucleic acid single linearcDNA/RNA NO YES 7 CCAATGCACC CAAGACAGCA GAAAG 25 26 base pairs nucleicacid single linear cDNA/RNA NO YES 8 TCCAATGCAC CCAAGACAGC AGAAAG 26 27base pairs nucleic acid single linear cDNA/RNA NO YES 9 CTCCAATGCACCCAAGACAG CAGAAAG 27 28 base pairs nucleic acid single linear cDNA/RNANO YES 10 GCTCCAATGC ACCCAAGACA GCAGAAAG 28 29 base pairs nucleic acidsingle linear cDNA/RNA NO YES 11 GGCTCCAATG CACCCAAGAC AGCAGAAAG 29 18base pairs nucleic acid single linear cDNA/RNA NO YES 12 CACCCAAGACAGCAGAAA 18 17 base pairs nucleic acid single linear cDNA/RNA NO YES 13CACCCAAGAC AGCAGAA 17 16 base pairs nucleic acid single linear cDNA/RNANO YES 14 CACCCAAGAC AGCAGA 16 21 base pairs nucleic acid single linearcDNA/RNA NO YES 15 CACCCAAGAC AGCAGAAAGT T 21 24 base pairs nucleic acidsingle linear cDNA/RNA NO YES 16 CACCCAAGAC AGCAGAAAGT TCAT 24 20 basepairs nucleic acid single linear cDNA NO YES 17 TCGCGCTCCC TCTCTCCGGC 2019 base pairs nucleic acid single linear cDNA NO YES 18 CATGGTTTCGGAGGGCGTC 19 21 base pairs nucleic acid single linear cDNA NO YES 19CAGCCTGGCT CACCGCCTTG G 21 21 base pairs nucleic acid single linear cDNANO NO 20 TCCGAAACCA TGAACTTTCT G 21 19 base pairs nucleic acid singlelinear cDNA NO NO 21 CTTTCTGCTG TCTTGGGTG 19 19 base pairs nucleic acidsingle linear cDNA NO YES 22 CTCCCAAGAC AGCAGAAAG 19 19 base pairsnucleic acid single linear cDNA NO YES 23 CTGCCAAGAC AGCAGAAAG 19 19base pairs nucleic acid single linear cDNA NO YES 24 CACCCAACTCTCCAGAAAG 19 19 base pairs nucleic acid single linear cDNA NO YES 25CACCCAAGAC AGCAGAATG 19 19 base pairs nucleic acid single linear cDNA NOYES 26 CACCCAAGAC AGCAGATTG 19

What is claimed is:
 1. A method of inhibiting vascular endothelialgrowth factor in tumor cells comprising the step of locallyadministering to said tumor cells, a therapeutically effective amount ofa synthetic oligonucleotide complementary to a human vascularendothelial growth factor target nucleic acid, whereby expression ofsaid vascular endothelial growth factor is inhibited, theoligonucleotide having a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,and SEQ ID NO:16.
 2. The method of claim 1, wherein the oligonucleotidehas a modification selected from the group consisting of analkylphosphonate, phosphorothioate, phosphorodithioate, phosphate ester,alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphatetriester, acetamidate, and carboxymethyl ester internucleotide linkage,and a combination thereof.
 3. The method of claim 2, wherein theoligonucleotide has at least one phosphorothioate internucleotidelinkage.
 4. The method of claim 3, wherein the oligonucleotide hasphosphorothioate internucleotide linkages.
 5. The method of claim 1,wherein the oligonucleotide has 2′-O-alkylated ribonucleotides.
 6. Themethod of claim 5, wherein the oligonucleotide comprises four or five2′-O-alkylated ribonucleotides at the 5′ terminus of theoligonucleotide.
 7. The method of claim 5, wherein the oligonucleotidecomprises four or five 2′-O-alkylated ribonucleotides at the 3′ terminusof the oligonucleotide.
 8. The method of claim 1, wherein theoligonucleotide is administered topically.